CN114908029B

CN114908029B - Construction and application of II-type grass carp reovirus VP6 recombinant lactobacillus

Info

Publication number: CN114908029B
Application number: CN202210427863.1A
Authority: CN
Inventors: 尹纪元; 王庆; 石存斌; 王楠; 李家豪; 王英英; 李莹莹; 张德锋; 莫绪兵
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2023-03-21
Anticipated expiration: 2042-04-22
Also published as: CN114908029A

Abstract

The invention discloses construction and application of II-type grass carp reovirus VP6 recombinant lactobacillus, wherein the recombinant lactobacillus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2022323. Relevant detection proves that the recombinant lactobacillus can directly present antigen to antigen presenting cells in a digestive tract immune system through oral immunization, can stimulate a fish body to simultaneously generate local mucosal immune response and systemic immune response reaction, and improve the fish body to generate specific and non-specific immune response reaction. The survival rate of gobiocypris rarus after immunization in the GCRV infection process is improved, the relative survival Rate (RPS) is 42.9%, and the feed of L.lactis pNZ8148-VP6 has good ecological prevention and control effect in the GCRV infection process.

Description

Construction and application of II-type grass carp reovirus VP6 recombinant lactobacillus

Technical Field

The invention belongs to the technical field of genetic engineering and molecular immunology, and particularly relates to construction and application of a type II grass carp reovirus VP6 recombinant lactic acid bacterium.

Background

Grass carp (Ctenophagogon idella) is the freshwater aquaculture fish with the highest yield in China and has an important economic position. However, grass Carp Hemorrhagic Disease (GCHD) with death rate as high as 80% frequently occurs, but no effective treatment is available at present. GCHD is caused by Grass Carp Reovirus (Grass Carp Reovirus, GCRV) infection, and epidemiological investigation results show that the genotype of the current main epidemic strain is GCRV-II type. Vaccination is the most effective method of protecting grass carps against GCRV infection.

The GCRV virus genome consists of 11 discontinuous double-stranded RNA segments with different sizes, and can code 12 proteins, including 7 proteins which are structural proteins and 5 proteins which are non-structural proteins. Among them, the structural protein VP1 is encoded by the GCRV S1 segment, and is presumed to have methyltransferase activity (methyl) during transcription of viral RNA and to be involved in the capping of viral mRNA replication, but the neutralizing activity of the antibody produced after immunization of rabbits is not high. The VP2 protein is encoded by the GCRV S2 segment and is presumed to have RNA polymerase activity. The VP3 protein is encoded by the GCRV S3 segment, is presumed to have nucleosidic hydrolase and RNA helicase activities, and is primarily involved in energy supply during viral transcription. After the grass carp is immunized by the recombinant baculovirus-assembled type II GCRV VP3, VP4 and NS38 particle-like viruses, immune protection can be generated. However, like VP2, VP3 does not produce neutralizing antibodies after immunization of rabbits. The type II GCRV VP4 proteins are all encoded by the S6 segment, are the main outer capsid proteins, participate in the transcription process of the viral genome and are related to the formation of viral inclusion bodies. Researches show that the bacillus subtilis oral vaccine and the DNA vaccine capable of expressing GCRV VP4 protein can be used for immunizing grass carp to generate specific antibodies and have a certain immune protection effect. The GCRV VP5 protein of type II is encoded by an S5 segment, has a reovirus M2 conserved structural domain in the structure, is related to the process of entering virus particles into host cells, and has nucleosidic hydrolase activity but no neutralizing activity of antibodies. The VP6 protein is used as a core protein of GCRV, can be connected with an inner capsid and an outer capsid, has good neutralization activity, can enable fish bodies to generate specific antibodies by oral administration or injection of vaccines, can effectively inhibit GCRV infection, and has higher similarity with delta 2 protein of mammal orthoreovirus shown by research. The VP7 protein and the incomplete VP5 protein can form a heterodimer to form an outer capsid of the GCRV, the protein cleavage effect is achieved, and the GCRV infection can be effectively resisted by immunizing the grass carp with the recombinant VP7 protein or DNA vaccine.

The vaccination is the most effective way for preventing and treating GCRV infection, and although the 'grass carp bleeding disease tissue homogenate inactivated vaccine' is effective, the risk of toxin dispersion caused by incomplete inactivation exists, and the protection rate is low, and the ideal protection effect can be obtained by repeated vaccination. The grass carp hemorrhagic disease attenuated live vaccine can obtain a good protection effect only by carrying out immunization once, but the attenuated vaccine has the risk of strong virulence. In addition, both the inactivated vaccine and the attenuated vaccine need to be injected for immunization to protect the grass carps, so that the operation is complicated, and the popularization and the use in fishery production are not facilitated.

The oral vaccine is prepared by encapsulating antigen or treating biological carrier, feeding bait containing immune preparation to make vaccine enter digestive tract mucosa of fish body, and stimulating intestinal mucosa immune system of fish body to generate immune response. The fish oral vaccination is not limited by time, place and fish size, is suitable for large population vaccination, is convenient to operate, saves time and labor, and is convenient to popularize and apply in large scale. However, the protein antigen has poor stability and low utilization rate in a water body environment, is easily degraded by protease in the digestive tract, and influences the immune protection effect. There is therefore a need for an oral formulation which can be effectively immunised.

Disclosure of Invention

The invention aims to construct recombinant lactobacillus capable of expressing GCRV VP6 protein by using a lactobacillus model strain as a vector. Then through oral gavage immunization gobiocyprisrarus, the resident condition of the recombination lactic acid bacteria in the intestinal tissue of back is confirmed through viable count, the protective effect of bacterin is evaluated through the expression level of the relevant gene of detection immunoreaction, serum specific antibody level and the immune protection rate to gobiocyprisrarus after attacking poison to the development of safe effectual II type GCRV probiotic oral vaccine provides the guarantee for the healthy breed of grass carp.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a recombinant lactic acid bacterium is provided, wherein the recombinant lactic acid bacterium comprises a gene sequence for encoding a VP6 of grass carp reovirus, and the amino acid sequence of the VP6 of grass carp reovirus is shown as SEQ ID No. 1.

In some embodiments of the invention, the gene sequence encoding VP6 of the grass carp reovirus is shown as SEQ ID No. 2.

In some embodiments of the invention, the recombinant lactic acid bacteria are constructed by introducing a recombinant plasmid carrying a gene sequence encoding for grass carp reovirus VP6 into the lactic acid bacteria.

In some embodiments of the present invention, the recombinant lactic acid bacteria are deposited at the chinese type culture collection, at the following: wuhan, china, the preservation number is CCTCC NO: m2022323, class name: lactococcus lactis pNZ8148-VP6 with preservation time of 2022, 03 and 28 days.

The lactobacillus is used as a carrier to prepare the ecological prevention and control product for grass carp hemorrhage, and the oral administration immunization is simple, convenient, safe and not easy to cause fish stress, and is suitable for the immunization of fishes with large water surfaces and small specifications. In addition, the lactobacillus has good biological safety, is a generally accepted food safety level microorganism, does not need protein expression post-treatment, simplifies the downstream link of bioengineering production, and greatly reduces the production cost. The lactobacillus is a symbiotic bacterium existing in an animal mucous membrane system, has certain tolerance to acid-base bile salt, can well protect carried antigen, and can continuously stimulate a fish mucous membrane immune system to secrete mucous membrane antibodies. Meanwhile, substances such as lactic acid and bacteriocin generated by metabolism of lactic acid bacteria have an obvious inhibiting effect on common pathogenic bacteria of aquaculture, gastrointestinal tract flora is adjusted by means of space occupation and the like to maintain gastrointestinal tract microecological balance, the growth of a fish body can be promoted, and the immunity of the fish body is improved. Simultaneously, the lactobacillus also has various probiotic functions: can inhibit the growth of pathogenic bacteria by secreting organic acids such as lactic acid and bacteriocin such as bacterial peptide; the immune function is enhanced by activating T, B lymphocytes, and the immune globulin and antibody levels are improved, so that the group immunity of fish is improved; synthesizing enzymes such as protease, lipase, cellulase and the like, improving the utilization rate of the feed, and synthesizing a plurality of B vitamins such as vitamin B1, B2, B6, nicotinic acid and the like; the lactobacillus has good stability, is resistant to the acid environment of the digestive tract, and can carry antigen protein to intestinal mucosa immune tissues, so that the fish body can obtain specific immune protection.

In a second aspect of the invention, the recombinant bacterium of the first aspect of the invention is provided for use in preventing and or treating diseases caused by grass carp reovirus.

In some embodiments of the invention, the disease is a bleeding disease.

In some embodiments of the invention, the product is a formulation.

In some embodiments of the invention, the formulation is an oral formulation.

In some embodiments of the invention, an adjuvant is included in the product.

In some embodiments of the invention, the product can be used as a microbial preparation for ecological prevention and control, can be used alone or added into feed or compounded with other components, and can be splashed into the environment to play a certain role in preventing and/or treating diseases; can also be used as vaccine, and for fish, the survival rate can be improved by oral administration, which is simple, effective and convenient.

In some embodiments of the present invention, the formulation is a tablet, a powder, a granule, a capsule, a sustained release formulation, or other common formulations.

In a third aspect of the invention, there is provided a product comprising the recombinant lactic acid bacterium of the first aspect of the invention.

In some embodiments of the invention, the product is a formulation.

In some embodiments of the invention, the formulation is an oral formulation.

In some embodiments of the invention, an adjuvant is included in the product.

The beneficial effects of the invention are:

the invention analyzes the functional domain and structure of GCRV VP6, selects the N-terminal coding gene of VP6, optimizes codons, constructs recombinant plasmid pNZ8148-VP6, and transfers lactobacillus L.lactis NZ9000 in an electric conversion mode to obtain recombinant lactobacillus, which is named as L.lactis pNZ8148-VP6. And determining successful display expression of the target protein in the recombinant bacteria by using Western blot and indirect ELISA technology. Carrying out oral immunization on experimental animals, collecting spleen, kidney and hindgut tissues, and determining the expression level of immune related genes in each tissue through qPCR (quantitative polymerase chain reaction), wherein the results show that the recombinant lactobacillus can stimulate different immune tissues of a fish body to generate immune response reaction through the oral immunization; the level of specific antibody IgM in serum is measured by ELISA, and the result shows that the recombinant lactic acid can stimulate the fish body to generate specific immune response reaction. At 42d after immunization, GCRV virulent virus is injected intraperitoneally, the relative survival Rate (RPS) is 42.9%, and L.lactis pNZ8148-VP6 has a good protection effect when being taken orally.

Drawings

FIG. 1 shows the functional domain and structural analysis of GCRV VP6. FIG. 1A is a GCRV VP6 functional domain analysis; FIG. 1B shows the prediction of the structure of each stage at the N-terminal of GCRV VP6.

FIG. 2 shows the PCR and restriction enzyme identification of the recombinant plasmid pNZ8148-VP6. The result of identifying the recombinant plasmid pNZ8148-VP6. And (A) PCR identification. M: DNA marker (DL 5000); lanes 1-3: a VP6 gene; (B) enzyme digestion identification. M: DNA standard (DL 5000); lane 1: nco I; lane 2: xba I; lane 3: double enzyme digestion; lane 4: pNZ8148-VP6 plasmid.

FIG. 3 is a recombinant lactobacillus GCRV VP6 protein fusion expression western-blot and analysis chart; m: a protein Marker; lane 1: l.lactis NZ9000; lane 2: l.lactis pNZ8148-VP6.

FIG. 4 is an analysis chart of indirect ELISA of recombinant lactobacillus GCRV VP6 protein fusion expression.

FIG. 5 shows the detection of serum-specific antibody levels by indirect ELISA; the level is expressed as the absorbance at 450 nm. Data are presented as mean ± standard deviation. The L.lactis pNZ8148-VP6 group was compared with the PBS group. Values with significant differences from the control group are indicated by asterisks (one-way anova, <0.05, <0.01, < 0.001).

FIG. 6 shows GCRV challenge protection assay after oral administration of immune recombinant lactic acid bacteria; FIG. 6A shows PCR-specific amplification of GCRV-infected fragments. Lane 1: negative control; lanes 2-7: a PBS group; lanes 8-13: lactis NZ9000 group; lanes 14-19: lactis pNZ8148-VP6 group; m: DNA marker (DL 5000); lane 20: GCRV positive control. FIG. 6B is a cumulative mortality curve of gobiocypris rarus after immunization after being detoxified by GCRV-HuNan 1307. Mortality was monitored daily for 14 days after challenge. * Indicating a significant difference in cumulative mortality at 14d in the immunized group compared to the PBS group (P < 0.05).

Fig. 7 is an analysis of the antigen retention capacity of recombinant lactobacillus in the intestinal tract of gobiocyprisrarus. And (3) counting colonies and detecting the residence condition of the recombinant lactobacillus in the intestinal tract by PCR. FIG. 7A shows the growth of L.lactis pNZ8148-VP6 in the intestinal tract of experimental fish on M17 agar plates resistant to chloramphenicol. Fig. 7B is the viable count of recombinant bacteria in the intestinal tract of gobiocypris rarus at the indicated time after immunization. Data represent means ± SEM of the trimaran. FIG. 7C shows the specific amplification of VP6 fragments by PCR of recombinant bacteria randomly picked from the plate. M: DNA marker (DL 5000), lanes 1-10: VP6 gene.

FIG. 8 is a graph of an immunoassay in the intestine after oral immunization of recombinant lactic acid bacterium L.lactis pNZ8148-VP6. FIG. 8A: IFN2, fig. 8B: IL-1 β, FIG. 8C: IRF7, fig. 8D: MHCII, fig. 8E: mx, fig. 8F: myD88, fig. 8G: NF-. Kappa.B, FIG. 8H: TLR3, fig. 8I: TLR5. The relative mRNA expression level of the immune related gene was calculated by using the 2- Δ Δ Ct method. According to the expression condition of the beta-actin gene, the mRNA level of each gene is normalized. Compared to the PBS group, significant differences (p < 0.05) and very significant differences (p < 0.01) were indicated.

FIG. 9 is a diagram of an immunoassay in the kidney after oral immunization of recombinant lactic acid bacterium L.lactis pNZ8148-VP6. FIG. 9A: IFN2, fig. 9B: IL-1 β, fig. 9C: IRF7, fig. 9D: MHCII, fig. 9E: mx, fig. 9F: myD88, fig. 9G: NF-. Kappa.B, FIG. 9H: TLR3, fig. 9I: TLR5. The relative mRNA expression level of the immune related gene is calculated by adopting a 2-delta-Ct method. According to the expression condition of the beta-actin gene, the mRNA level of each gene is normalized. Compared to the PBS group, significant differences (p < 0.05) and very significant differences (p < 0.01) were indicated.

FIG. 10 is a graph of an immunoassay in the spleen after oral immunization with recombinant lactic acid bacterium L.lactis pNZ8148-VP6. FIG. 10A: IFN2, fig. 10B: IL-1 β, fig. 10C: IRF7, fig. 10D: MHCII, fig. 10E: mx, fig. 10F: myD88, fig. 10G: NF-. Kappa.B, FIG. 10H: TLR3, fig. 10I: TLR5. The relative mRNA expression level of the immune related gene is calculated by adopting a 2-delta-Ct method. According to the expression condition of the beta-actin gene, the mRNA level of each gene is normalized. Compared to the PBS group, significant differences (p < 0.05) and very significant differences (p < 0.01) were indicated.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Experimental animals: gobiocypris rarus is donated by aquatic organisms of Chinese academy of sciences, and has a body length of 4 +/-0.5 cm.

Bacteria and strains: the gene II type grass carp reovirus is separated and stored in an aquatic disease and immunity research room of the Zhujiang aquatic research institute, and the separation method comprises the following steps: PSF cells are adopted for isolated culture in a laboratory, and the specific method comprises the following steps: the virus is inoculated on a compact single-layer PSF cell, the cell is adsorbed for 2 hours at 28 ℃, an M199 culture medium containing 5 percent fetal bovine serum is added for culture after adsorption liquid is removed, the cell is continuously cultured for 5 to 7 days at 28 ℃ and the virus is harvested by repeated freeze thawing, and the specific method can be seen in research on grass carp reovirus molecular epidemiology, whole genome sequence and epidemic strain inactivated vaccine (D) Zeng Weiwei, southern agriculture university.

The main reagents are as follows: pNZ1848 expression vector, escherichia coli MC1061, lactococcus lactis NZ9000, gobiocypris rarus IgM monoclonal antibody were prepared in the laboratory by the method described in Weiwei Zeng, et al.development of a VP38 recombinant protein-based index ELISA for detection of antibodies against peptides GCRV II. JFD (2018) 41; the first article is an experiment for preparing an antibody, a grass carp antibody is prepared, and the second article discloses that the grass carp antibody can be used in gobiocypris rarus, so that the antibody is also used for carrying out related detection. Trizol Reagent, primeScript ^TM RT reagent Kit, takara Nco I, xba I restriction enzyme, takara Ex

Genomic DNA extraction kits, premix Ex TaqTM (Probe qPCR) and component Cell Preparation Kit were purchased from Baygian technology Inc., SYBR Green Pro Taq HS Premix qPCR was purchased from Yu Aike R Biotech Inc., LB broth and M17 broth medium were purchased from Beijing land bridge technology Inc., chloramphenicol was purchased from Beijing Solaley technology Inc., nisin was purchased from Shanghai Merland Biotechnology Inc., and SDS-PAGE and Western blot-related reagents were purchased from Suzhou Xinsai Biotechnology Inc.

Example 1GCRV VP6 bioinformatics analysis

According to the nucleotide sequence of the VP6 gene of the GCRVHuNan 1307 strain virus gene II (GenBank accession number is KU 254574.1) submitted in NCBI, the amino acid sequence of the nucleotide sequence is obtained through an Expasy online amino acid translation website (https:// web.

The SMART tool website was used to predict specific functional domains of the GCRVVP6 protein.

The protein secondary structure was analyzed by the PSIpred-MEMAT 3 online analysis server.

The three-level structure of the GCRV VP6 protein is compared and folded through SWISS-MODEL software on an ExPASy website, and a space construction map of the protein is simulated or constructed.

The transmembrane region of VP6 was analyzed using TMHMM2.0 online server (https:// www.cbs.dtu.dk/services/TMHMM).

FIG. 1A is a GCRV VP6 functional domain analysis, and the VP6 protein comprises two functional domains, located at the N-and C-termini of the protein, both formed from separate trimers. Trimer of the N-terminal fiber tail is formed without participation of ATP or chaperonin and is directly arranged into three coils to be fixed on virus particles in a spiral manner; the C-terminal globular head interacts with protein receptors, and its post-translational assembly is associated with the phosphorylation of Hsp 90. In addition, a small region was found at the N-terminal tail to bind sialic acid on the surface of target cells, presumably to be associated with promoting apoptosis. Therefore, the important role of the N-terminal of VP6 in the virus infection process can be a potential antigen protein with good immunogenicity.

FIG. 1B shows the prediction of the N-terminal structure of GCRV VP6. The N-terminus of VP6 contains two secondary structures, alpha-helix and random coil, respectively; the N end of VP6 is a simple tertiary structure formed by connecting 5 alpha-helices together through several random curls with different lengths, and the N end of VP6 has no transmembrane region, no higher-level structure or complex modification processing, and is relatively suitable for expression in a prokaryotic expression system.

Example 2 preparation of recombinant plasmid expressing GCRV VP6

And (3) synthesizing and identifying a recombinant expression vector pNZ8148-VP6.

Coli MC1061 competent cells were prepared according to the kit instructions:

(1) The low temperature centrifuge is precooled at 4 ℃ before the test, and the whole test is careful to keep the low temperature.

(2) The MC1061 glycerol strain stored at-80 ℃ was streaked out onto LB solid plates and incubated at 37 ℃ overnight.

(3) A single colony was picked up in LB medium and cultured overnight with shaking at 37 ℃.

(4) The bacterial solution was inoculated in LB medium at a ratio of 1.

(5) Subpackaging the bacterial liquid, centrifuging 1mL bacterial liquid per tube at 4 ℃ and 4000r/min for 5min, and discarding the supernatant.

(6) Add Solution A (precooled on ice) 100. Mu.L per tube and mix gently.

(7) Centrifuging at 4000r/min for 5min at 4 ℃, and discarding the supernatant.

(8) Add Solution B (precooled on ice) 100. Mu.L per tube and mix gently.

(9) Storing at-80 deg.C.

According to a gene sequence of an expression vector pNZ8148, selecting two enzyme cleavage sites of Nco I and Xba I to insert a GCRV VP 6N end gene into the gene, adding a section of His label (consisting of 6 to 10 histidine residues) before Xba I, and optimizing the advanced codon of the inserted sequence according to the expression preference of lactic acid bacteria, wherein the optimization is realized according to the preference of lactococcus lactis on coding amino acid codons in a protein expression process and the whole GC content in the inserted fragment, partial bases are adjusted, the codon optimization is more favorable for the expression of the inserted fragment in the lactococcus lactis according to the degeneracy principle of codons, and the codon optimization is realized in an artificial synthesis way.

The sequence of the His tag is as follows: CATCATCATCATCACCAC (SEQ ID NO. 3).

The nucleotide sequence of the GCRV VP6 is as follows: ATAGTATCTGAAGCATATCAATCCATCGCAATGGG ACCGCTCACCT TGCAAGATGGTTACTATCGAGCACTTAGCGTGATTACCTTAATTTACCT TGCGTCGCTGACAGGCCGCCTTGGTCCTGACAGAACTTATTACGGGTTTTATGTTCAATT CCCAAAAAAGCGTAAATTTGAAGATTTAGGGTATTTTGCTTATAATGCTGATGGAAGAAA TGTTGCTGTTTTACAATCAATCAATGCCTATATTTATTGTGCAAGTCCTGATTGGCAATATT CTTGTGCTCTTTATTATCTCCATGTCTTGTCAGCTTTGAGTCTTTCTTGGACTGACCCAGT TGGAATGATTGACGGTTTTAGTTGCGTAAATCAATTTACAGATGTTCCAGGTTGGTCAGC GACAAATCGAGCCTTACATACACATTCATTCAACTGGTTTAATTTACTTGAGGATGCTATT GATACTTTGGTCGCCCGCCGTTATTGGACTAACGCAGAAGGACAGGCAATTCGTCAAGA ATGGACAGCAGCTAGAGATCGTTGGCGTGTTATTATGGATGCAACTCGTGATGAAGATGA CTTAGTGGTTTTTAGGACACCAGATGATTGTCGTCGTCGGCTAAAACCTTATGGTGACAA TAATTGGACGAGAGCT (SEQ ID NO. 2);

the amino acid sequence of the GCRV VP6 is as follows: MGPLTLQDGYYRALSVITLIYLASLTGRLGPDRTY YGFYVQFPKKRKF EDLGYFAYNADGRNVAVLQSINAYIYCASPDWQYSCALYYLHVLSAL SLSWTDPVGMIDGFSCVNQFTDVPGWSATNRALHTHSFNWFNLLEDAIDTLVARRYWTNA EGQAIRQEWTAARDRWRVIMDATRDEDDLVVFRTPDDCRRRLKPYGDNNWTR (SEQ ID NO. 1).

The recombinant expression vector pNZ8148-VP6 was synthesized by Kinsley Biotechnology, inc. The received plasmid lyophilized powder was diluted for subsequent handling according to the instructions.

The synthesized recombinant expression vector pNZ8148-VP6 is transformed into E.coli MC1061 competent cells by the following steps:

(1) The water bath was preheated to 42 ℃ before testing.

(2) Taking out the competent cells stored in a refrigerator at-80 deg.C, thawing on ice for 30min, adding 1ng of pNZ8148-VP6 plasmid into a clean bench, mixing gently, and standing in ice for 25min.

(3) Heating in 42 deg.C water bath for 45s, and rapidly standing in ice for 5min.

(4) Adding M17 culture medium into the test tube, and resuscitating for 1h at 37 ℃ by shaking.

(5) Centrifuging at 1000r/min for 1min, and discarding part of supernatant.

(6) The remaining bacterial solution was mixed and uniformly spread on LB solid plate (containing 5. Mu.g/mL chloramphenicol).

(7) The culture was carried out overnight at 37 ℃.

And (3) selecting the monoclonal antibody, inoculating the monoclonal antibody into a LB (LB) culture medium containing chloramphenicol resistance, performing shake culture at 37 ℃ at 200r/min for 12h, extracting plasmids, performing enzyme digestion verification and PCR (polymerase chain reaction) identification, and sending the plasmids to a sequencing company for sequencing. The PCR and enzyme digestion identification result shows that the recombinant plasmid pNZ8148-VP6 is successfully constructed (figure 2).

Example 3 preparation of recombinant lactic acid bacteria expressing GCRV VP6

Preparing L.lactis NZ9000 competent cells, and storing the prepared competent cells to-80 ℃. After extracting pNZ8148-VP6 plasmid, electrically transforming the plasmid into the competent cells, wherein the transformation steps are as follows:

(1) Before the test, the electric rotary cup and the recovery medium (containing 0.5M sucrose and 0.02M MgCL) were added ₂ 0.002M CaCL ₂ M17 liquid medium).

(2) L.lactis NZ9000 competent cells were thawed in ice for 10min, 1. Mu.g of plasmid was added to a clean bench, gently mixed, and allowed to stand on ice for 10min.

(3) And transferring the mixture into a pre-cooling electric rotating cup with the diameter of 2mm, and wiping water drops outside the electric rotating cup to avoid cup explosion.

(4) Click quickly, the shock condition is: 25000V,5ms.

(5) Quickly adding 900 μ L of precooling recovery culture medium, uniformly mixing, placing on ice for 10min, anaerobically culturing at 30 ℃ for 4h, centrifuging at 3500r/min for 1min, and discarding 800 μ L of supernatant.

(6) And (3) blowing and suspending the residual bacteria liquid, uniformly coating the bacteria liquid on an M17 solid plate containing 5 mu g/mL chloramphenicol, and performing anaerobic standing overnight culture at the temperature of 30 ℃. Single colonies were extracted into M17 liquid medium containing 10. Mu.g/mL of chloramphenicol and subjected to static culture at 30 ℃. The correctly identified recombinant lactic acid bacteria were named L.lactis pNZ8148-VP6. The recombinant strain is preserved in China center for type culture Collection for 28 days in 2022 years; the preservation number is CCTCC NO: m2022323, class name: lactococcus lactis pNZ8148-VP6.

Expression of recombinant lactic acid bacteria. The transformant containing the positive recombinant plasmid pNZ8148-VP6 was selected, inoculated into 10mL of M17 liquid medium (containing 10. Mu.g/mLCm), and subjected to anaerobic static culture at 30 ℃ overnight. Taking culture bacteria liquid according to the proportion of 1:50 proportion is inoculated in 300mL of M17 liquid medium containing 10 mug/mL of chloramphenicol, anaerobic static culture is carried out at 30 ℃ for 4h to enable the liquid medium to be in logarithmic growth phase, 1ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL and 100ng/mL of inducer Nisin are respectively added, induction is carried out at 30 ℃ for 4h, and non-induced recombinant lactococcus lactis is used as a control.

Immunoblot (Western-blot) analysis of the expression products. Centrifuging the samples before and after induction at 4 ℃ and 12000r/min for 15min, discarding the supernatant, suspending with 6mL of 1 XPBS solution, and performing ultrasonic disruption with the disruption power of 200w, working for 2s, and resting for 5s for 30min. Adding SDS gel sample buffer proportionally, mixing, boiling for 10min, and performing protein electrophoresis analysis on 12% SDS-PAGE. After SDS-PAGE electrophoresis, proteins on the gel were transferred to NC membrane, blocked in 5% skimmed milk powder, and incubated overnight at 4 ℃ or 2h at 37 ℃. Washing 3 times 5min each time by adding PBST (PBS +0.05% Tween-20). The monoclonal antibody of the VP6 protein is used as a primary antibody, and the incubation is carried out at 4 ℃ overnight (more than or equal to 15 h). PBST was added and washed 3 times for 5min each. HRP-labeled goat anti-mouse IgG was added as a secondary antibody and incubated at room temperature for 1h. PBST was added and washed 3 times for 5min each. Visualization was performed according to the DAB visualization kit instructions and the results were observed. After induction, the recombinant lactic acid bacteria showed a clear response at the expected position of the expressed protein band, while the control group did not (FIG. 3).

Indirect ELISA analysis of the expression products. And respectively taking the induced L.lactis pNZ8148-VP6 subjected to ultrasonic wall breaking and the complete thallus as antigen coating 96-hole ELISA reaction plates (enzyme-labeled plates), taking L.lactis NZ9000 as a negative control, coating each hole by 100 mu L at 4 ℃ overnight. PBST was washed 3 times for 5min each. Each well was sealed by adding 300. Mu.L of skim milk powder containing 5%. And (4) cleaning, and synchronizing step 2. mu.L of diluted monoclonal antibody to VP6 protein was added to each well and incubated overnight at 4 ℃. Cleaning and synchronizing the step 2. mu.L of diluted HRP-labeled goat anti-mouse IgG was added to each well and incubated at room temperature for 1h. Cleaning and synchronizing the step 2. Adding dye solution according to the instruction, developing for 5-10min at 37 deg.C in dark, adding 50 μ L stop solution, and measuring OD450nm value with enzyme labeling apparatus. All data are expressed as mean ± sem. Data were analyzed by Student's T test, and P <0.05 was considered statistically significant. The OD450nm values of the L.lactis pNZ8148-VP6 groups are all significantly different from those of the control group (p < 0.05). Meanwhile, the results of the broken thallus coating and the thallus direct coating have no significant difference (p is more than or equal to 0.05). It was revealed that VP6 protein was carried on the surface of the cells (FIG. 4).

Example 4 fusion expression of GCRV VP6 protein oral immunoprotection evaluation

Healthy gobiocypris rarus is randomly divided into 3 groups, each group has 100 tails and is respectively a PBS blank control group, an L.lactis NZ9000 empty bacteria control group and an L.lactis pNZ8148-VP6 immune group. The immunization mode is intragastric administration and oral administration, the immunization is carried out once every 2 weeks for 2 times, and each time of continuous infusionStomach 3d, immune dose 10 uL 2X 10 ⁹ CFU/mL/fish/d.

And (3) randomly taking 5 gobiocypris rarus from each group after immunization, collecting spleen, kidney and hindgut tissues, detecting the expression level of the intracellular immune related cytokines, collecting serum samples at 3w and 6w after immunization, and collecting 10 fishes per group for detecting the level of serum IgM specific antibodies. The samples were collected and stored at-80 ℃.

Sera from immunized and control fish (10 fish per group) were collected at 3w and 6w post-immunization, respectively, for antibody titer detection. Gobiocypris rarus adopts a tail-off blood-taking method, and the specific operation is as follows: the gobiocypris rarus is used for collecting blood after the tail is broken. Standing the collected blood at 4 deg.C overnight, centrifuging at 5000r/min for 5min, collecting upper layer serum, and storing at-20 deg.C.

The purified GCRV virus was diluted with ELISA coating buffer to a final protein concentration of 100ng/ml, 50. Mu.L of the diluted virus was added to each well of the microplate, 3 replicates of each sample were set, and coated overnight at 4 ℃. PBST was washed 3 times for 5min each. Add 250. Mu.L of 5% skim milk powder to each well and block for 2h at 37 ℃. And (4) cleaning, and synchronizing step 2. Serum samples were diluted 200-fold using PBS solution, 50. Mu.L per well and incubated for 1h at 37 ℃. Cleaning and synchronizing the step 2. Add 100. Mu.L of IgM monoclonal antibody (1 diluted 10000) per well and incubate for 1h at 37 ℃. Cleaning and synchronizing the step 2. mu.L of HRP-labeled goat anti-mouse IgG antibody was added to each well and incubated at room temperature for 45min. Cleaning and synchronizing the step 2. Adding dye solution according to the instruction, developing at 37 deg.C in dark for 5-10min, adding stop solution, and measuring OD450nm with enzyme labeling apparatus. All data are expressed as mean ± sem. Data were analyzed by Student's t test, and P <0.05 was considered statistically significant. The results show that oral administration of lactobacillus expressing VP6 can induce fish to produce specific antibodies (fig. 5).

And (5) evaluating a challenge test. At 42d after immunization, 20 mu L of GCRV Hunan1307 virulent strain with 10LD50 concentration (LD 50 is 10-5.25LD50/20 mu L) is injected into the abdominal cavity of each fish, and the challenge fish is kept to be raised in a water temperature environment of 28-30 ℃. And (5) monitoring all experimental fishes within 14 days after the challenge, recording morbidity and mortality, and calculating relative protection rate. The results show that the survival rate of experimental fish in GCRV infection can be improved by oral administration of the immune recombinant lactobacillus, and the relative protection rate of L.lactis pNZ8148-VP6 is 42.9% (figure 6).

Example 5 evaluation of immunoregulatory Effect of recombinant lactic acid bacteria orally administered grass carp

And (4) evaluating the intestinal retention capacity of the recombinant lactobacillus fishes. Intestinal tissue was viable count after collection at 3h, 6h, 12h, 24h, 48h and 72h after the first immunization to determine if recombinant lactic acid bacteria could reside for a long time in the hindgut site for antigen presentation. 500 μ L of 1 XPBS solution was added to each sample and homogenated for 1, 10 ¹ 、10 ² 、10 ³ 、10 ⁴ 、10 ⁵ Dilution by fold, 200. Mu.L of each sample was pipetted and spread onto M17 solid plates containing 5. Mu.g/mL chloramphenicol, and incubated overnight at 28 ℃ under standing for colony counting. The results show that the recombinant lactic acid bacteria can stay in the intestinal tract of fish for more than 72 hours after oral administration (fig. 7).

And (3) detecting the immunity enhancement effect of the recombinant lactobacillus on the fish after oral immunization. Respectively randomly taking 5 gobiocypris rarus from each group at 7d, 14d, 21d and 28d after immunization, collecting spleen, kidney and hindgut tissues, extracting total RNA of each tissue, then carrying out reverse transcription, taking beta-actin as an internal reference gene, taking the obtained cDNA as a template, and determining the relative expression amounts of TLR3, TLR5, myD88, NF-kappa B, IFN, mx, IRF7, MHCII and IL-1 beta in each tissue by qRT-PCR. The primer sequences of the respective genes are shown in Table 1, and the primer sequences were synthesized by the above-mentioned sequencer. The experiment was performed in triplicate. The qRT-PCR reaction system is as follows: 10 μ L of 2 XSSYBR Green Taq HS Premix, 0.4 μ L each of primers, 0.4 μ L ROX,2 μ L cDNA template and plus ddH ₂ Supplementing the total amount of O to 20 mu L, performing pre-denaturation at 95 ℃ for 5min, and then circulating: denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 45 s; the cycle was 35 times. The results were analyzed using the 2- Δ Δ Ct algorithm. All data are mean ± sem. Data were analyzed by Student's t test, P<0.05 was considered statistically significant. The result shows that the recombinant lactobacillus can be directly identified and presented by antigen presenting cells in fish intestinal mucosa immune tissues after oral immunizationAn antigen; processed by lymphocytes in immune organs such as spleen and kidney, and elicited an immune response (fig. 8-10).

TABLE 1 primer sequences in fluorescent quantitative PCR

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

<110> Zhujiang aquatic research institute of Chinese aquatic science research institute

<120> construction and application of II-type grass carp reovirus VP6 recombinant lactic acid bacteria

<130>

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 207

<212> PRT

<213> GCRV VP6

<400> 1

Met Gly Pro Leu Thr Leu Gln Asp Gly Tyr Tyr Arg Ala Leu Ser Val

1 5 10 15

Ile Thr Leu Ile Tyr Leu Ala Ser Leu Thr Gly Arg Leu Gly Pro Asp

20 25 30

Arg Thr Tyr Tyr Gly Phe Tyr Val Gln Phe Pro Lys Lys Arg Lys Phe

35 40 45

Glu Asp Leu Gly Tyr Phe Ala Tyr Asn Ala Asp Gly Arg Asn Val Ala

50 55 60

Val Leu Gln Ser Ile Asn Ala Tyr Ile Tyr Cys Ala Ser Pro Asp Trp

65 70 75 80

Gln Tyr Ser Cys Ala Leu Tyr Tyr Leu His Val Leu Ser Ala Leu Ser

85 90 95

Leu Ser Trp Thr Asp Pro Val Gly Met Ile Asp Gly Phe Ser Cys Val

100 105 110

Asn Gln Phe Thr Asp Val Pro Gly Trp Ser Ala Thr Asn Arg Ala Leu

115 120 125

His Thr His Ser Phe Asn Trp Phe Asn Leu Leu Glu Asp Ala Ile Asp

130 135 140

Thr Leu Val Ala Arg Arg Tyr Trp Thr Asn Ala Glu Gly Gln Ala Ile

145 150 155 160

Arg Gln Glu Trp Thr Ala Ala Arg Asp Arg Trp Arg Val Ile Met Asp

165 170 175

Ala Thr Arg Asp Glu Asp Asp Leu Val Val Phe Arg Thr Pro Asp Asp

180 185 190

Cys Arg Arg Arg Leu Lys Pro Tyr Gly Asp Asn Asn Trp Thr Arg

195 200 205

<210> 2

<211> 654

<212> DNA

<213> GCRV VP6

<400> 2

atagtatctg aagcatatca atccatcgca atgggaccgc tcaccttgca agatggttac 60

tatcgagcac ttagcgtgat taccttaatt taccttgcgt cgctgacagg ccgccttggt 120

cctgacagaa cttattacgg gttttatgtt caattcccaa aaaagcgtaa atttgaagat 180

ttagggtatt ttgcttataa tgctgatgga agaaatgttg ctgttttaca atcaatcaat 240

gcctatattt attgtgcaag tcctgattgg caatattctt gtgctcttta ttatctccat 300

gtcttgtcag ctttgagtct ttcttggact gacccagttg gaatgattga cggttttagt 360

tgcgtaaatc aatttacaga tgttccaggt tggtcagcga caaatcgagc cttacataca 420

cattcattca actggtttaa tttacttgag gatgctattg atactttggt cgcccgccgt 480

tattggacta acgcagaagg acaggcaatt cgtcaagaat ggacagcagc tagagatcgt 540

tggcgtgtta ttatggatgc aactcgtgat gaagatgact tagtggtttt taggacacca 600

gatgattgtc gtcgtcggct aaaaccttat ggtgacaata attggacgag agct 654

<210> 3

<211> 18

<212> DNA

<213> His

<400> 3

catcatcatc atcaccac 18

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

ctatgttggt gacgaggctc a 21

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

cccagttggt gacaataccg 20

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence

<400> 6

ttggtagagg ctaatgcg 18

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence

<400> 7

aatggaggac aaccgaga 18

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence

<400> 8

aagggtgctt ggagataa 18

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<400> 9

ttgaaagtcc cagatgaa 18

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

ggtggtaatt tccgatga 18

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<400> 11

gtagacaaca gggataagg 19

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<400> 12

aactcagtca ggctccattg c 21

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence

<400> 13

gacagtgctc tccgtctttc c 21

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

acagtcaagc aggaggagga 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<400> 15

tcactggcgc tgtctgtatc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<400> 16

gacacgctgt cctctggtat 20

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<400> 17

cagtttcttt gtttggctct g 21

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence

<400> 18

ccaagagcag agccagtt 18

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence

<400> 19

tagggcgtcc caaagtag 18

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<400> 20

aatgacgacg gcacttacaa 20

<210> 21

<211> 18

<212> DNA

<213> Artificial sequence

<400> 21

actcccagca gccccaga 18

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

tgatgagatg gactgccctg 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

tgtccgtctc tcagcgtcac 20

Claims

1. The recombinant lactobacillus is preserved in China center for type culture Collection with the preservation time of 2022 years, 3 months and 28 days, and the preservation number is CCTCC NO: m2022323, class name: lactococcus lactis pNZ8148-VP6;

the recombinant lactic acid bacteria comprise a gene sequence for coding the grass carp reovirus VP6, and the amino acid sequence of the grass carp reovirus VP6 is shown as SEQ ID NO. 1;

the recombinant lactobacillus is constructed by introducing recombinant plasmids carrying gene sequences encoding grass carp reovirus VP6 into lactobacillus.

2. Use of the recombinant lactic acid bacteria of claim 1 for the preparation of a product for the prevention and/or treatment of hemorrhagic disease in grass carp.

3. Use according to claim 2, wherein the product is a formulation.

4. The use according to claim 3, wherein the formulation is an oral formulation.

5. The use according to claim 2, wherein the product further comprises an adjuvant.

6. A product comprising the recombinant lactic acid bacterium of claim 1.

7. The product of claim 6, wherein the product is a formulation.

8. The product of claim 7, wherein the formulation is an oral formulation.

9. The product of claim 6, further comprising an adjuvant.